Plants as biofactories for the production of proteins is a technology that is being employed by a number of groups for edible vaccines, pharmaceuticals and industrial enzymes (Hood and Jilka, 1999; Hood and Howard, 1999). Pharmaceutical and vaccine production in plants has several advantages in that the material contains no contaminating organisms and can be directly consumed (Hood and Jilka, 1999; Hood and Howard, 1999). Production of industrial enzymes in plants provides the possibility of considerably reduced production costs, the benefit of recovered costs through sale of by products, easier transportation and reduced chance of contamination.
Over-expression of an industrial enzyme in a transgenic plant requires quite high expression levels to make the system economically viable, a condition that has been achieved for several proteins, e.g. the diagnostic protein, avidin (Hood et al. 1997) and laccase (WO 00/20615). Using plants as biofactories for industrial enzyme production provides considerable advantages over traditional methods of such enzyme production, since plants provide easier transport and cost savings, but also can be far more readily produced in large quantities than when produced in bacteria or fungi, for example, allowing for even further increases in the amount of enzyme which may be produced.
Achieving high levels of enzyme production in plants is impacted by several factors, such as location of expression of the enzyme within specific tissues and within particular subcellular compartments to insulate the plant tissues from the activity of the enzymes. Thus, in WO 00/20615, it is discussed that preferentially directing expression to the seed of the plant and also to plant cell wall tissue and to the endoplasmic reticulum of the plant cell is advantageous in increasing enzyme protein production.
In addition to increased concentrations of enzymes, it is desired that the enzymes exhibit high activity. While some enzymes depend for activity only on their structure as proteins, others also require one or more non-protein components, termed cofactors. The cofactors may be a metal ion or an organic molecule called a coenzyme and some enzymes require both. Cofactors are generally stable to heat, where most enzyme proteins lose activity on heating. The term holoenzyme is used to refer to the catalytically active enzyme-cofactor complex. When the cofactor is absent, the protein, which is catalytically inactive by itself, is called an apoenzyme. Transitional metal ions are important cofactors in enzymatic transformation of nonmineral substances in anabolic and catabolic processes within plant cells. Therefore, the presence of such transitional metal ions may be important in providing an active enzyme.
Plants produce many of these cofactors as an essential element of their vegetative growth process in considerable amounts. Thus, one would presume that the plant would supply adequate quantities of the metal ion needed to produce active enzyme. For example, about four atoms of copper are needed for each molecule of laccase in order to produce active laccase enzyme. A person skilled in the art would expect there would be more than enough copper available since there is a considerable amount of copper for enzyme uptake in the plant. In fact, there is about 20 ppm copper in normal corn tissues, which would be sufficient to support laccase accumulation at much greater than 5ng/mg seed weight (see Table 1).
Thus there is about a thousand times more copper in the corn plant than is necessary to support laccase expression at 5 ng/mg. There should be more than enough available for production of active laccase when it is produced in a plant. Instead, the inventors have found this is not the situation. Unless such transition metals are added over and above what is pesent in plants, the amount of active enzyme is reduced. By providing such cofactors during plant development and/or during or after protein extraction from the plant tissue, the amount of active enzyme is increased, at times greater than ten fold. This is particularly surprising, since attempts to add the metal cofactor copper to laccase fungal expression systems have not met with success in improving activation levels of the enzyme.
Additionally, the inventors have found that by incubating the metal and enzyme while controlling the temperature during incubation, either during extraction or after, it increases the recovery of active protein by such possible mechanisms as refolding and stabilization of the protein or reoxidation of the transition metal. Negative salt ions added during or after extraction of the enzyme with the metal further aid in improving recovery of active enzyme.
Optimal conditions have also been discovered by the inventors for improved recovery of laccase using the copper cofactor.
The invention relates to the discovery by the inventors that while transgenic plants expressing enzymes contain considerable quantities of transitional metal cofactors needed for certain enzyme activation, it is necessary to provide additional metal ions in order to increase recovery of active enzyme from plants.
Therefore it is an object of the invention to provide a process for increasing recovery of active enzyme from a plant where that enzyme requires a transitional metal cofactor, by providing additional metal cofactor to the enzyme, either during plant development, during extraction of the enzyme from the plant, following extraction of the enzyme from the plant, or during all three phases.
A further object of the invention is to increase recovery of active enzyme from a plant in which a transitional metal cofactor has been added by further adding a negative salt ion.
Yet another object of the invention is increasing recovery of active laccase which is produced by a plant having a nucleotide sequence encoding laccase by providing additional copper to such laccase enzymes.
An object of the invention is a method of increasing recovery of active laccase which is produced in a plant having a nucleotide sequence encoding laccase by adding a negative salt ion to the laccase enzyme, preferably where the ion is chloride.
A further object of the invention is a method of increasing recovery of active organophosphate hydrolase which is produced by a plant having a nucleotide sequence encoding organophosphate hydrolase by providing additional transitional metals such as zinc, nickel, cobalt or manganese to such organophosphate hydrolase enzymes.
An object of the invention is a method of increasing recovery of active ogranophosphate hydrolase enzymes by adding a negative salt ion to the enzyme, preferably where the ion is chloride.
The invention further has as an objective incubating the metal and enzyme while controlling temperature of the incubation. The temperature that provides improved recovery will vary with time of incubation but practical considerations indicate that recovery is improved when the incubation with the metal is for up to several weeks when at 4xc2x0 C., preferably up to several days when incubated at room temperature (20xc2x0-27xc2x0 C.); preferably at room temperature up to 37xc2x0 C. for about 20 to 60 minutes when a negative salt ion is added; and up to three hours at 50xc2x0 C. Still another object of the invention is to provide for optimal yield of active laccase produced in plants by using a solution to extract the laccase having a copper salt solution of 0.05mM to 1M copper, preferably 1 mM to 100 mM copper, more preferably 10 to 30 mM copper